Strain growth and development
D. discoideum cells of strain AX2 were grown either with Klebsiella aerogenes on SM agar plates or axenically in liquid nutrient medium  in shaking suspension at 160 rpm at 21°C. gxcDD- cells were cultivated in axenic medium containing 3.5 μg/ml blasticidin (ICN Biochemicals, OH). To analyse development, cells were grown axenically to a density of 2–3 × 106/ml, washed twice in Soerensen phosphate buffer (17 mM Na-K phosphate, pH 6.0) and 5 × 107 cells were plated on phosphate agar plates. The streaming pattern was studied by allowing 1 ml of 5 × 106 cells/ml to settle on a well of a NUNC six-well plate and observed at 3 min intervals with a Leica DM-IL inverse microscope.
Generation of gxcDD- cells and molecular cloning
For disruption of the GxcDD gene in AX2 cells, a GxcDD gene replacement vector was constructed using the plasmid pBSBsrΔBam . A 1.1-kb 5' fragment coding for the CH domain and the IQ motifs was PCR amplified using the forward primer 5-ATGCAACCCAAAGATTATATG-3' and reverse primer 5-ACTATTGTAATGGATGAT-3' and a 1.0-kb 3' fragment was PCR amplified using forward primer 5'-TTAATGAGTTGTATGAGAAGA-3' and reverse primer 5'-TGTGCAGAATGTGGAGCATCA-3' from AX2 genomic DNA. The PCR products obtained were cloned into pGEM-T Easy cloning vector (Promega GmbH). The gene fragments were released and cloned into pBSBsrΔBam. The resulting replacement vector was linearised by digesting with PvuII and transformed into AX2 by electroporation. Transformants were selected in nutrient medium containing blasticidin (3.5 μg/ml). Independent clones were screened for the disruption of the GxcDD gene by PCR using genomic DNA, Southern blotting and western blot analysis. For Southern blot analysis a probe encompassing nucleotides 3807–4860 was used.
For expression of the CH domain of GxcDD fused to green fluorescent protein (GFP) at the N-terminus, a 0.4-kb fragment encoding the first 134 residues of GxcDD was amplified from AX2 cDNA and cloned into pBsr-GFP [37, 38]. A 1-kb fragment encoding residues 395–707 containing the RacGEF domain was amplified and cloned in pGEX-4T1 (Amersham Biosciences) for expression in E. coli. The C-terminal 1-kb fragment encoding residues 1269–1619 containing the ArfGAP-PH tandem was amplified and cloned into pBsr-GFP for expression in AX2 cells and into pGEX-4T3 for expression in E. coli. Generation of strains expressing RacF1, RacG, RacH fused to GFP has been described elsewhere [12, 13, 39]. For expression of Rac1a, RacA (GTPase domain), RacB, RacC, RacD, RacE, RacI and RacJ fused to the C-terminus of GFP, PCR was performed on corresponding cDNAs. PCR products were cloned into pDEX-GFP  and the sequence verified. These vectors were introduced into AX2 cells and clones were selected by visual inspection under a fluorescent microscope.
Generation of polyclonal antibodies specific for GxcDD
Polyclonal antibodies specific for GxcDD were obtained by immunising female white New Zealand rabbits with GST-ArfGAP-PH (100 μg/animal; Pineda Antikörper-Service, Berlin), followed by two boosts of 100 μg each at two weeks intervals. The antiserum was affinity purified by incubating with GST-ArfGAP-PH bound glutathione-sepharose beads.
For separating membrane and cytosolic fractions cells were washed in Soerensen buffer and resuspended at a density of 1 × 108 cells/ml in MES buffer (20 mM MES, pH 6.5, 1 mM EDTA, 250 mM sucrose supplemented with protease inhibitor cocktail (Sigma)). Cells were lysed by sonication and membrane and cytosolic fractions separated by centrifugation at 100,000 g for 30 min at 4°C.
Triton X-100 was used for preparing cytoskeletal fractions. Cells were washed as before and resuspended in Soerensen buffer at a density of 5 × 107 cells/ml and 300 μl of cell suspension were lysed using an equal volume of TIC buffer (2% Triton X-100, 20 mM KCl, 20 mM imidazol, pH 7.0, 20 mM EGTA, 4 mM NaN3) and incubated on ice for 10 min followed by incubation at RT for 10 min. The insoluble cytoskeleton fraction was separated by centrifugation at 10,000 g for 4 min. Supernatant and pellet fractions were subjected to western blot analysis.
GST pulldown assays
GST-RacGEF and GST-ArfGAP-PH were expressed in E. coli and bound to glutathione-sepharose beads. For interaction of GxcDD with Rac proteins, 4 × 107 AX2 cells expressing Dictyostelium Racs as GFP fusions were lysed in lysis buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 1 mM NaF, 0.5 mM Na3VO4, 1 mM DTT, supplemented with protease inhibitors (Sigma)) and incubated with equal amounts of GST-RacGEF bound beads for 3 hrs at 4°C. Beads were washed with wash buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA). The eluate of the pulldown was immunoblotted and the Rac protein detected using a GFP specific monoclonal antibody . Cells expressing only GFP were used as a control. For interaction studies of the ArfGAP-PH domain GST-ArfGAP-PH bound to glutathione-sepharose beads was incubated with AX2 cell lysates. For interaction studies of the CH domain with RacGEF and ArfGAP-PH domain, glutathione-sepharose beads bound to GST fused RacGEF and ArfGAP-PH tandem were incubated with AX2 cells expressing GFP-CH domain. Pulldown eluates were immunoblotted and probed with GFP specific monoclonal antibody. GST bound beads were used as a control.
Immunoflorescence was done by fixing cells using cold methanol for 10 min followed by staining for actin using actin-specific monoclonal antibody Act1-7  and Cy3 conjugated secondary antibody. Live cell imaging of fluorescent cells in suspension or during phagocytosis of TRITC labelled yeast particles was done by laser scanning confocal microscopy essentially as described . Capillary chemotaxis was done as described using a Leica DM-IL inverse microscope and analyzed using DIAS .
Monoclonal antibodies recognizing α-actinin , contact site A , pspA , comitin  and GFP  were used for western blotting.
F-actin levels upon cAMP stimulation were determined as described . Briefly, aggregation competent cells resuspended at 2 × 107 cells/ml were stimulated with 1 μM cAMP and 50 μl samples were taken at various time points. Samples were immediately lysed in lysis buffer (3.7% formaldehyde, 0.1% Triton X-100, 0.25 μM TRITC-phalloidin in 20 mM potassium phosphate, 10 mM PIPES, pH 6.8, 5 mM EGTA, 2 mM MgCl2) and stained for 1 hr and centrifuged at 10,000 g for 5 min. Pellets were extracted with 1 ml methanol overnight and fluorescence (540/565) measured in a fluorimeter.
For crosslinking experiments GST-ArfGAP-PH was thrombin cleaved on glutathione-sepharose beads and the purified protein subjected to dialysis against PBS, pH 7.4, for 6 hrs at 4°C. To equal amount of protein increasing amount of glutaraldehyde (0–0.1% v/v) was added. The final reaction volume was 40 μl. Crosslinking was carried out at 4°C for 30 min. The reaction was stopped by addition of 5 μl 1 M glycine. Samples were subjected to imunoblotting.
Phosphoinositides binding was performed by a dot blot assay. 200 pmoles of each phophoinositide (PtdIns(3,4)P2, PtdIns(4,5)P2, PtdIns(3,4,5)P3) were spotted on a PVDF membrane and incubated with ArfGAP-PH domain. Protein bound to lipids was detected using polyclonal GxcDD antibodies.
For cell aggregation in suspension gxcDD- and AX2 cells were allowed to starve in Soerensen buffer at a density of 1 × 107 cells/ml and samples were withdrawn at the indicated times. The percentage of aggregated cells was determined by measuring the OD600.
For northern blot analysis total RNA was isolated from AX2 cells of different developmental stages as described previously and separated in agarose gels containing 6% formaldehyde [49, 50]. The blot was probed with a fragment derived from the 3' end of the GxcDD cDNA (nt 3807–4860)